Focal adhesions are specialized structures in the plasma membrane involved in the adhesion of a cell to a substrate, such as the extracellular matrix (ECM). Focal adhesions form the connection between an extracellular substrate and the cytoskeleton, and affect such functions as cell shape, cell motility and cell proliferation. Transmembrane integrin molecules form the basis of focal adhesions. Upon ligand binding, integrins cluster in the plane of the plasma membrane. Cytoskeletal linker proteins such as the actin binding proteins alpha-actinin, talin, tensin, vinculin, paxillin, and filamin are recruited to the clustering site. Key regulatory proteins, such as Rho and Ras family proteins, focal adhesion kinase, and Src family members are also recruited. These events lead to the reorganization of actin filaments and the formation of stress fibers. These intracellular rearrangements promote further integrin-ECM interactions and integrin clustering. Thus, integrins mediate aggregation of protein complexes on both the cytosolic and extracellular faces of the plasma membrane, leading to the assembly of the focal adhesion. Many signal transduction responses are mediated via various adhesion complex proteins, including Src, FAY, paxillin, and tensin (for a review, see Yamada, K. M. and B. Geiger, (1997) Curr. Opin. Cell Biol. 9.76-85.). Src activity plays a key role in the formation of tumors and the development of cancers. In particular, the increased Src activity can be an early event in cancer development, as evidenced in pre-malignant lesions and adenomas. Biosensors that can directly or indirectly detect a change in Src activity provide a mechanism for the early detection of neoplastic cells. To introduce a biosensor into a cell, there are mainly two methods: gene-based and protein-based. The gene-based method requires the introduction of a genetically encoded biosensor into a cell. It takes time for cells to incorporate the biosensors into their genomes and produce encoded proteins through transcription and translation. The low efficiency for transfection methods in general will result in the failure of the identification of non-transfected cancerous cells.
Protein-based methods are similarly limited. The plasma membrane of the cell generally prevents the trespass of polar or large-molecular-weight (>500 Da) molecules. Traditional methods of introducing proteins into the cells involve fusing the desired proteins to receptor ligands or packaging them to liposomal carriers. However, these methods are labor-intensive and usually result in the trapping of desired proteins in endocytic organelles and hence not able to express their proper functions.
Accordingly, methods and compositions for the efficient introduction and detection of biosensors in a living cell are needed.